Snakes on the Brain: Essays on Snakes, Science, and Society. Essay 1. The Brown Snake, The Taipan, and The Paradox.

Snakes on the Brain: Essays on Snakes, Science, and Society # 1

I often wonder what exactly about snakes makes them such polarizing figures throughout human history. We tend to react in extremes when it comes to these often maligned creatures, and both human-parties, whether the fascinated or the fearful, view the other as either misguided or perhaps certifiable. To this day it remains surprising to some that fans of snakes, such as me and many other perfectly rational folk, even exist, let alone work with and study these stunning, adaptive, mysterious beasts.

These essays are my menial attempt to bridge some of the gap between the fascinated and the fearful, while exploring some aspect of science or natural history. While I make no claim of authority, I will say that I aim to provide as much reasoning and references for my views wherever necessary throughout. Where I fall short, I apologize and request your unyielding criticism, dear reader, as both punishment for the current and polish for future essays.

Essay 1. The Brown Snake, The Taipan, and The Paradox

I love fools’ experiments. I am always making them.” – Charles Darwin

As a zoology student in Brisbane, Australia, whenever classmates would ask me for study tips, they were often disappointed to find I rarely start at the beginning of anything. My ideal study method is to dive into the middle of a subject at an interesting juncture and read widely around this nugget of intrigue, often at the temporary expense of the subject at large. As my own interest in a subject blossoms, so does my curiosity for the overall details of whatever system I am learning about, then perhaps I am ready to tackle the early introductory subject matter (not to mention lecture slides on general housekeeping and assessment dates, the horrors of which no undergraduate need reminisce on). Without further delay, let us then dive headlong into the world of snakes, and perhaps throughout come to a greater understanding of this bizarre collection of animals. We should I believe, for interest’s sake, start with a discussion on a peculiar aspect of the venoms of two of Australia’s deadliest snakes; one the most common venomous snake we have, the other the longest and arguably most formidable.

When it comes to the venoms of various animals, neurotoxins are typically the most complex and the most lethal. While a typical “neurotoxin” is simply any substance harmful to nerve tissues, the protein based neurotoxins developed for hunting, defense, or competition, in various groups of organisms are among the most dangerous substances on the planet. In fact, the most toxic substance currently known to humanity is a neurotoxic excretion from the Clostridium botulinum bacteria and its relatives, known as Botulinum Toxin (BTX), or Botox to those in the cosmetic industry, blocking cholinergic neuromuscular junctions, which prevents motor fiber stimulation and can cause death by paralysis of the respiratory system (1). If it seems ludicrous to be injecting this bacterial neurotoxic protein, the most dangerous substance known to humanity, into one’s cheeks to lift and tighten the skin, well, I may agree with you however we must both somewhat acknowledge that the science of using sub-lethal doses of toxic substances for therapeutic purposes is well established. As for the various neurotoxins of the animal world, how very few, relative to the expansive available variety, have been recklessly injected into people’s cheeks for vanity and/or science? Think of what we might learn with but a little bravery!

Of course, this is foolishness. I simply hope to impress upon the reader two things; first, the efficacy and usefulness of biological neurotoxins for human medical science, and second, the potential untapped resources for biodiscovery in the diversity of our planet’s venomous fauna (2, interested readers would do well to check out this 2014 review by Alan L Harvey for the journal Toxicon). The study of toxic substances, venoms included, is called toxicology. The integration of chemistry, biology, and medicine is what makes toxicology so fascinating, with many toxicologists working broadly in all three fields. While not restricted to fauna (environmental toxicology, for example, is an extremely useful tool for societies who wish to avoid the pitfalls of environmental contaminants, poisons and catastrophes. Toxinology is the more specific study of animal, plant, and microbial toxins, but the umbrella term should serve us fine), the Toxicologists who study animal venoms are a dedicated bunch, often travelling far and wide to find some elusive subject to “milk” for venom, which is stored on ice and rushed back to the laboratory as fast as the local means of transport will allow.

While this sounds all rather adventurous, we will instead focus on the recent work of toxicologists on something more familiar. Here on Australia’s east coast, you don’t have to go far to find our first subject. The Eastern Brown Snake (Pseudonaja textilis), also rightfully known as the Common Brown Snake (hereon referred to as “EB”), is a relatively large, alert, fast-moving snake of the family Elapidae, the front fanged snakes including cobras and coral snakes (3). While often regarded as dangerous or “aggressive”, in reality it is quick to avoid people and will flee rather than fight. When cornered or agitated, however, the EB’s defensive display is formidable. The fore-body is raised in an S-shape, displaying the often vibrant reddish-brown “belly spots” on the ventral surface, with the bright pink mouth open. They are capable of moving in this position using the posterior part of the body, keeping the S-shape raised as they approach a would-be attacker (4). This display is so effective that anecdotal reports of being “chased by a brown snake” often arise from those fleeing in terror. Needless to say, once you’re outside the snakes perceived threat perimeter, the defensive posture collapses and they resume looking for shelter, but I’m certain the story of the Epic Chase continues over cold beverages.

It is only the truly unlucky or foolhardy who get bitten by Australian snakes, with most bites caused by accidentally treading on snakes, followed by those actively trying to harm or capture them, and the EB is no different in this regard (5,6, with more on snake bite fatalities in Australia). However its venom contains potent neurotoxins, procoagulents, and more nasty surprises for any biological system it encounters. Based on the LD50 (volume needed to kill, on average, 50% of a population of lab mice within a given time period) it is the second most venomous snake on land, however it is important to remember that this value is not based on human physiology, something to bear in mind as we introduce another formidable serpent (7).

Our next subject, the Coastal Taipan (Oxyuranus scutellatus, hereon referred to as “CT”), has possibly the most feared reputation of all Australian snakes. Found from east to north Australia and across to Papua New Guinea, these large, fast moving elapids are highly venomous, perhaps a consequence of their strict diet of warm blooded mammals, with the rather challenging prey of bandicoots as a favourite (8). In fact, there was a time not long ago, the 1960s I believe, when a confirmed Taipan bite came with a 100% mortality rate (9). It is also the longest venomous snake in Australia, beat out in weight only by the Mulga or King Brown Snake (a member of the genus Pseudechis rather than Pseudonaja, thus not a true “Brown” snake, but the name King Brown now has a mythical quality to some Australians, and if a name makes the foe that much more menacing at the retelling…), and arguably the fastest and most agile of our large elapids (8). While in possession of a similar cocktail of chemical weaponry, which I’m delighted to say we shall soon discuss in length, the venom delivery system in the CT is phenomenal, with 13mm long fangs on a large, wide hinged jaw for consuming prey as large as bandicoots (I suppose analogous in size to a small rabbit). The CT is also known to flee readily, in fact they can be darned hard to find, I can count on one hand the occasions I’ve seen them in the wild myself, even as a reptile obsessed geek who searched for wildlife almost constantly (thankfully with little or no supervision or guidance, and thus my numerous injuries and near misses). Unlike the EB however, the defensive posture is much lower and less static, lashing out in multiple strikes rather unpredictably, often inflicting multiple bites (10).

Beautiful creatures, no? I suppose some would disagree, however the aesthetic appreciation of snakes can wait for another time. Why are we discussing these two snakes, other than for their apparent badassitude in terms of death and venom? The reason I bring up these two snakes, in relation to toxicology specifically, is due to a rather interesting medical mystery in the patient outcomes of human victims of EB and CT bites. The so called “Brown Snake Paradox”, coined in 2002, refers to the puzzling phenomenon that an EB bite rarely results in systemic neurotoxicity, while for a CT bite neurotoxicity is the major feature, manifesting as loss of muscle control, including the muscles controlling the respiratory and circulatory system (11). While both snakes have a variety of procoagulent toxins which interfere with natural clotting processes, causing internal bleeding and hemorrhages, this seems to be the main effect of EB venom. The main clinical effect of brown snake bites is VICC (Venom Induced Consumption Coagulopathy, say it with me now…) where the powerful procoagulent venom molecules consume all the clotting factors available in the blood (fibrinogens etc.). This inhibits clotting ability, causes microangiomas, and a host of other complications. It kind of like Stigmata on your everything.

The surprising part comes when you compare the LD50 of these snakes. When comparing the whole venom of these snakes, containing both pre- and post-synaptic neurotoxins, as well as myotoxins and procoagulants, the CT comes in as the 3rd most venomous snake in Australia, but it is beaten out by the humble, “common brown”! In fact, the EB ranks as the second most venomous land snake in the world, beat out only by the Inland Taipan (Oxyuranus microlepidotus) (9). Given this information, shouldn’t we suspect that an EB bite would result in severe neuropathy?

The Brown Snake Paradox was a medically significant conundrum for years. When it comes to treating a snakebite victim, the variety of complications is wide and varied, but the possible complications are still somewhat defined by the type of venom and its activity in the body (12). Neurotoxins often cause respiratory distress and require assisted breathing, while procoagulents may necessitate clotting factor replacement, or dialysis in cases of renal failure. Early treatment is of course the best course for survival and recovery, so we would prefer to understand the biological activity of these venoms and treat them as necessary, rather than inferring from symptoms what treatments are required. Whether this supposedly powerful textilotoxin causes human neurotoxicity is important for the whole snakebite scenario “Chain-Of-Survival”. While CPR and assisted breathing may certainly be necessary, if the main systematic threat is loss of clotting factors, then, brothers and sisters, you better hope there are some clotting factors on the way for you! Anti-venom therapy can only do so much, and the damage done by the venom on the way to hospital is still a health hazard which may require treatment. Knowing which treatments work for which species of snake, from which regions of Australia (enjoyably, snake venoms can vary in their bioactivity regionally, see 13), and early treatment are key.

So we have a paradox; the highly “neurotoxic” EB which causes little to no neurotoxicity. Could perhaps its lethality be limited to other venom functions? How actively neurotoxic are they? These questions and more are usually where the Toxicologists step in. To be fair, they’ve been here the whole time and are the only reason any of us actually know what an LD50 is! Thanks to some phenomenal work by Carmel M. Barber et al (2012) the Brown Snake Paradox is effectively solved, if only in how and perhaps not the why. Lets us first consider this newly illuminated how, and perhaps we might speculate on the unknown why.

The neurotoxins of elapid snakes can be classified into two groups major: postsynaptic neurotoxins and presynaptic neurotoxins (14). In 1979, the presynaptic neurotoxins of our stories two protagonists were identified by separate research groups, taipotoxin from Taipans (15), and textilotoxin from the EB (16) (again, the Latin name is P. textilis, thus the name of the venom).  When tested on mice, textilotoxin has a much lower LD50 than taipotoxin, meaning it requires significantly less of the venom to kill the same amount of a test population. It was thus determined that textilotoxin was the largest and most potent neurotoxin isolated. How then did the team of Carmel M. Barber and co. defy this claim in 2012? As many venomous snake fanatics will tell you, LD50s alone are not to be taken as the Gospel of Snake Venom Toxicity (thou shalt not assume LD50s apply to all fauna?). What works in mice may not work in humans, but more significantly, the LD50 test itself is based on a whole organism test within a certain time limit, and “death within 12 hours or it’s all gravy” fails to take into account the amount of internal damage done to tissues and organs within those 12 hours. Damage which may suddenly result in further complications after the stopwatch for the LD50 test has finished. If you want to compare the neurotoxicity of two venoms, a more precise, direct method is needed.

This is precisely what Barber et al. achieved a few years ago (11). When new investigative methods arise, old prejudices must die like so many lab mice. Or in this case, lab chickens. Aside from extracting and comparing the amounts of the toxins, the two were compared on a Chick biventer cervicis nerve-muscle (CBCNM) preparation, a technique from the sixties that can help determine whether a toxin in pre- or post-synaptic in action, and help quantify it’s direct affect on nerve signal transduction from a motor neuron to a muscle fibre. From a euthanized chicken, a motor neuron and connected muscle tissue are delicately dissected and, in a medium, hooked up to a small electrical generator. Electrical signals are then used to simulate signals from the brain, causing the muscle to twitch at a defined interval. To compare potencies of the venoms, they compared the time taken to block the twitches from nerve stimulation by 90%. This value, t90, was much, much lower in taipotoxin, indicating it actually has a much greater effect on the nervous system then textilotoxin. This finding, along with the lower concentrations of textilotoxin found in EB venom suggests a resolution to the paradox; the more potent Taipotoxin makes a greater proportion of CT venom, thus imbuing their bite with a highly neurotoxic effect, while the textilotoxin on the EB has a comparatively lower effect on the nervous  system, and is present in lower concentrations. Other venom components, such as the prothrombin activator (a procoagulent which makes up over a third of EB venom) frequently cause EB victims to suffer from VICC and other complications, rather than paralysis. The paradox is, for now, fairly well dead.

So, we now accept that textilotoxin has a reduced activity on nerve function. Where does this leave us? Is there a ready explanation for this reduced neurotoxic activity? After all, we can assume from the wide variety of neurotoxic snakes that such venoms are historically very effective and a successful survival tool. Why abandon neurotoxicity?

While no solid hypotheses have yet been subjected to experiment, several potential theories come to mind. First, despite the lower neurotoxicity to the CBCNM preparation, rodents still seem rather susceptible to EB neurotoxins, and perhaps some accumulated differences in our receptor sites renders humans, and perhaps chickens, with some immunity. Second, textilotoxin is but one (albeit large and complex) neurotoxin in the EB cocktail, in fact we have completely ignored the post-synaptic neurotoxins, which act at the neuromuscular junction and more rapidly than our current focus, the pre-synaptic neurotoxins. The cocktail of post-synaptic neurotoxins may hide their own secrets! (For example, see this investigation of post synaptic neurotoxins between putative subspecies of CT, 17)

Thirdly, perhaps this reduction is a result of selective pressures acting on the venom composition of EB populations. It has already been shown that there is some variation in venom between geographically distant populations due to, it is thought, differences in dietary preference and availability (13). Venom genes evolve rapidly (18, 19), as do their upstream control genes, and perhaps with this rapid mutation rate the genes for non-neurotoxic venom components are, for some reason, achieving greater success. I can imagine a scenario where prey populations are under such strong pressure to evolve immune defenses to the newly evolved neurotoxins, and neurotoxins in turn evolve greater potency, and so on until building powerful, large, complex neurotoxins is the norm, and native prey items require strong immunity. Such evolutionary arms races are commonplace, and venoms are the result of this chemical arms race over thousands of generations.

But during such an arms race for neurotoxicity specifically, perhaps the selection pressures to evolve immunity to other newly evolved proteins, such as the prothrombin activators in EB venom, are relaxed somewhat in comparison (though not enough to disappear completely as they are still a minor component of many venoms) (20). If then, perhaps, a mutation in the control regions for a gene which is responsible for producing such procoagulents etc. causes an increase in production to well above what the prey’s immune system has historically encountered, the owner of said mutation might have greater success and leave more offspring. This of course increases the frequency of this successful mutation in the population. Given time, with snakes now relying on procoagulent effects rather than neurotoxicity, the gene for textilotoxin might be allowed to accumulate mutations at a near-neutral rate, with no functional purging of less effective variants since it is of little significance to catching dinner. The rifle gets relegated to the back of the attic to collect dust among the rest of the now retired hunting tools, replaced by the compound carbon-fiber hunting bow.

Speculation can be dangerous. The human mind’s ability to project ourselves into a virtual future have been proven flawed repeatedly, and we are prone to false conclusions, unconscious biases, and personal preferences which make fools of us all. As such, the above “whys” are to be taken with much more salt than the “hows” of the Brown Snake Paradox. Nonetheless, without imagination, we might never conceive the true nature of complex, time dependent processes, like poisoning for example. Imagination, conceptualization, and my-best-educated-guess are fundamental tools of science. After such fanciful thought wanderings, however, science puts its imagination on a scale. One consistently incredible facet of science to me is how scientists can imagine new ways to verify previously imagined realities. Perhaps, thanks to imaginative and dedicated toxicologists, we will one day know why the Brown Snake Paradox exists.

New methods lead to new discovery. Very few of us would consider that in our lifetime medically significant new information on the venom of the common, humble brown snake would come to light. Those deep in the field however know differently, and my admiration for those that continue to explore how the chemistry of life interacts with itself is limitless. Taking a deeper look into the neurotoxicity of these deadly and beautiful snakes, the Eastern Brown Snake and the Coastal Taipan, is a challenge. They are agile snakes, alert and fast, very capable of defending themselves if push comes to shove. Still, I like to imagine the smiles on the faces of some snake handlers and toxicologists in the field, trying to explain to baffled onlookers or locals how the venom from the gloriously deadly animal in his catch bag had a date with destiny, to be applied to electrically stimulated chicken flesh in order to save human lives.

Sometimes it’s a lot of fun, and very weird, to dig a little deeper.

Ciao,
Janne
www.snakeoutbrisbane.com.au

Your SnakeOut Brisbane.

Essay #2 can be found here.

References
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